Establishment of human corneal epithelial organoids for ex vivo modelling dry eye disease.

Dry eye disease (DED) is a growing public health concern affecting millions of people worldwide and causing ocular discomfort and visual disturbance. Developing its therapeutic drugs based on animal models suffer from interspecies differences and poor prediction of human trials. Here, we established long-term 3D human corneal epithelial organoids, which recapitulated the cell lineages and gene expression signature of the human corneal epithelium. Organoids can be regulated to differentiate ex vivo, but the addition of FGF10 inhibits this process. In the hyperosmolar-induced DED organoid model, the release of inflammatory factors increased, resulting in damage to the stemness of stem cells and a decrease in functional mucin 1 protein. Furthermore, we found that the organoids could mimic clinical drug treatment responses, suggesting that corneal epithelial organoids are promising candidates for establishing a drug testing platform ex vivo. In summary, we established a functional, long-term 3D human epithelial organoid that may serve as an ex vivo model for studying the functional regulation and disease modelling.

Membrane Fusion-Mediated Loading of Therapeutic siRNA into Exosome for Tissue-Specific Application.

Tissue-specific delivery of oligonucleotide therapeutics beyond the liver remains a key challenge in nucleic acid drug development. To address this issue, exploiting exosomes as a novel carrier has emerged as a promising approach for efficient nucleic acid drug delivery. However, current exosome-based delivery systems still face multiple hurdles in their clinical applications. Herein, this work presents a strategy for constructing a hybrid exosome vehicle (HEV) through a DNA zipper-mediated membrane fusion approach for tissue-specific siRNA delivery. As a proof-of-concept, this work successfully fuses a liposome encapsulating anti-NFKBIZ siRNAs with corneal epithelium cell (CEC)-derived exosomes to form a HEV construct for the treatment of dry eye disease (DED). With homing characteristics inherited from exosomes, the siRNA-bearing HEV can target its parent cells and efficiently deliver the siRNA payloads to the cornea. Subsequently, the NFKBIZ gene silencing significantly reduces pro-inflammatory cytokine secretions from the ocular surface, reshapes its inflammatory microenvironment, and ultimately achieves an excellent therapeutic outcome in a DED mouse model. As a versatile platform, this hybrid exosome with targeting capability and designed therapeutic siRNAs may hold great potential in various disease treatments.

Reversible Assembly of Proteins and Phenolic Polymers for Intracellular Protein Delivery with Serum Stability.

The design of intracellular delivery systems for protein drugs remains a challenge due to limited delivery efficacy and serum stability. Herein, we propose a reversible assembly strategy to assemble cargo proteins and phenolic polymers into stable nanoparticles for this purpose using a heterobifunctional adaptor (2-formylbenzeneboronic acid). The adaptor is easily decorated on cargo proteins via iminoboronate chemistry and further conjugates with catechol-bearing polymers to form nanoparticles via boronate diester linkages. The nanoparticles exhibit excellent serum stability in culture media but rapidly release the cargo proteins triggered by lysosomal acidity and GSH after endocytosis. In a proof-of-concept animal model, the strategy successfully transports superoxide dismutase to retina via intravitreal injection and efficiently ameliorates the oxidative stress and cellular damage in the retina induced by ischemia-reperfusion (I/R) with minimal adverse effects. The reversible assembly strategy represents a robust and efficient method to develop serum-stable systems for the intracellular delivery of biomacromolecules.

Framework Nucleic Acids Combined with 3D Hybridization Chain Reaction Amplifiers for Monitoring Multiple Human Tear Cytokines.

Existing tear sensors are difficult to perform multiplexed assays due to the minute amounts of biomolecules in tears and the tiny volume of tears. Herein, the authors leverage DNA tetrahedral frameworks (DTFs) modified on the wireless portable electrodes to effectively capture 3D hybridization chain reaction (HCR) amplifiers for automatic and sensitive monitoring of multiple cytokines in human tears. The developed sensors allow the sensitive determination of various dry eye syndrome (DES)-associated cytokines in human tears with the limit of detection down to 0.1 pg mL-1, consuming as little as 3 mL of tear fluid. Double-blind testing of clinical DES samples using the developed sensor and commercial ELISA shows no significant difference between them. Compared with single-biomarker diagnosis, the diagnostic accuracy of this sensor based on multiple biomarkers has improved by ≈16%. The developed system offers the potential for tear sensors to enable personalized and accurate diagnosis of various ocular diseases.

A Nucleic Acid-Based LYTAC Plus Platform to Simultaneously Mediate Disease-Driven Protein Downregulation.

Protein degradation techniques, such as proteolysis-targeting chimeras (PROTACs) and lysosome-targeting chimeras (LYTACs), have emerged as promising therapeutic strategies for the treatment of diseases. However, the efficacy of current protein degradation methods still needs to be improved to address the complex mechanisms underlying diseases. Herein, a LYTAC Plus hydrogel engineered is proposed by nucleic acid self-assembly, which integrates a gene silencing motif into a LYTAC construct to enhance its therapeutic potential. As a proof-of-concept study, vascular endothelial growth factor receptor (VEGFR)-binding peptides and mannose-6 phosphate (M6P) moieties into a self-assembled nucleic acid hydrogel are introduced, enabling its LYTAC capability. Small interference RNAs (siRNAs) is then employed that target the angiopoietin-2 (ANG-2) gene as cross-linkers for hydrogel formation, giving the final LYTAC Plus hydrogel gene silencing ability. With dual functionalities, the LYTAC Plus hydrogel demonstrated effectiveness in simultaneously reducing the levels of VEGFR-2 and ANG-2 both in vitro and in vivo, as well as in improving therapeutic outcomes in treating neovascular age-related macular degeneration in a mouse model. As a general material platform, the LYTAC Plus hydrogel may possess great potential for the treatment of various diseases and warrant further investigation.

Factors affecting long-term changes of meibomian gland in MGD patients.

PURPOSE: To explore the long-term course of patients with meibomian gland dysfunction (MGD), and to analyse potential factors affecting the recovery of meibomian gland (MG) dropout. METHODS: Seventy-nine MGD patients (79 eyes) aged 36.03±15.78 years old who underwent more than one year of follow-up were enrolled in this retrospective study. Corneal fluorescein staining (CFS), tear meniscus height (TMH), noninvasive breakup time (NIBUT), and noncontact meibography at baseline and last visit were collected and analysed. Then an automatic MG analyzer was used to measure the morphological and functional parameters of MGs, including their area ratio (AR), tortuosity index (TI), and signal index (SI). The patients whose AR increased by more than 5% were defined as MG improvement, and AR decreased by more than 5% was MG worsening. RESULTS: A total of 79 patients (79 eyes) were assessed with at least 1-year of follow-up. More than 1/3 of MGD patients (27 eyes, 34.2%) underwent MG improvement, and 30.4% of MGs became worsened. Age (P=0.002), gender (P<0.001), IPL treatment (P=0.013), the change of CFS (P=0.0015), and the recovery of SI (P=0.035) showed significant differences among different recovery groups. Age(P<0.001), female sex (P=0.003), ΔCFS (P<0.001), AR at baseline (P<0.001) were negative correlation with AR recovery, and the change of SI (P=0.003) and IPL treatment (P=0.003) had a positive correlation with it. Among them, age (P=0.038), the change of CFS (P=0.004), and AR at baseline (P=0.007) were confirmed as negatively correlated factors predicting the long-term change of the MG. CONCLUSION: Although the MGD treatment has continued for more than 1 year, only 34.2% of MGD patients were observed to undergo MG improvement. Younger patients and patients with better CFS recovery seem to have more opportunities to improve their MGs.

R-(+)-WIN55212-2 protects pericytes from ischemic damage and restores retinal microcirculatory patency after ischemia/reperfusion injury.

BACKGROUND AND PURPOSE: Cannabinoids are vasoactive substances that act as key regulators of arterial tone in the blood vessels supplying peripheral tissues and the central nervous system. This study aimed to investigate the potential of R-(+)-WIN55212-2 (WIN), a cannabinoid receptor 1 agonist (CB1), as a treatment for retinal ischemia/reperfusion (I/R) injury. EXPERIMENTAL APPROACH: Male Wistar rats were subjected to retinal I/R injury by increasing intraocular pressure in the anterior chamber. The rats were randomly divided into four groups: normal control, I/R, vehicle (pre-treated with dimethyl sulfoxide [DMSO] via intraperitoneal injection), and experimental (pre-treated with WIN at a dose of 1 ml/kg via intraperitoneal injection). The rats were sacrificed at different time points of reperfusion (1 hour, 3 hours, 6 hours, and 1 day) after inducing retinal I/R injury, and their retinas were collected for analysis. Oxygen-glucose deprived/reperfusion (OGD/R) was performed by initially perfusing the retinas with oxygenated artificial cerebrospinal fluid (ACSF), then switching to an OGD solution to simulate ischemia, followed by another perfusion with ACSF. Pericyte contraction and the "no-reflow" phenomenon were observed using infrared differential interference contrast (IR-DIC) microscopy and immunohistochemistry. Western blot, enzyme-linked immunosorbent assay (ELISA), and nitric oxide (NO) detection were used to explore the potential mechanism. KEY RESULTS: In both the OGD/R and I/R models, retinal pericytes exhibited persistent contraction even after reperfusion. The ability of WIN to regulate the tone of retinal pericytes and capillaries was specifically blocked by the BKCa inhibitor iberiotoxin (100 nM). WIN demonstrated a protective effect against retinal I/R injury by preserving blood flow in vessels containing pericytes. Pretreatment with WIN alleviated the persistent contraction and apoptosis of retinal pericytes in I/R-induced rats, accompanied by a reduction in intracellular calcium ion (Ca2+) concentration. The expression of CB1 decreased in a time-dependent manner in the I/R group. After I/R injury, endothelium-derived nitric oxide (eNOS) levels were reduced at all time points, which was successfully reversed by WIN therapy except for the 1 day group. Additionally, the downregulation of cyclic guanosine monophosphate (cGMP) and BKCa expression at 3 hours, 6 hours, and 1 day after I/R injury was restored by pretreatment of WIN. CONCLUSIONS & IMPLICATIONS: WIN exerted its protective effects on retinal I/R injury by inhibiting the contraction and apoptosis of pericytes through the CB1-eNOS-cGMP-BKCa signaling pathway, thus ameliorated the occlusion of retinal capillaries.

Alpha 7-nicotinic cholinoceptor regulation of pericyte-containing retinal capillaries.

BACKGROUND AND PURPOSE: Local blood flow regulation relies on the coordination between neurons and pericyte-containing capillaries. Pericyte relaxation and contraction are influenced by vasoactive substances and regulated by neurotransmitters. α7 nicotinic acetylcholine receptors (α7-nAChRs), involved in the regulation of vascular function and inhibitory γ-aminobutyric acid (GABA) systems, have neuroprotective effects against CNS diseases. Although α7-nAChRs are found throughout the retina, their contribution to the retinal capillary tone remains unknown. Here, we investigated the neurovascular coupling mechanism underlying α7-nAChR-mediated retinal capillary tone regulation. EXPERIMENTAL APPROACH: Changes in capillary diameter and pericyte transverse diameter during drug perfusion were observed using differential interference contrast (DIC) microscopy, to help elucidate signalling pathways underlying α7-nAChR-mediated regulation of capillary blood flow at the whole retinal level. Patch clamp technique was used to investigate α7-nAChR-mediated regulation of the GABA synaptic circuit. Immunofluorescence was used to explore the expression of α7-nAChRs and GABA receptors. KEY RESULTS: Activating α7-nAChRs on the endothelial cell membrane caused perinuclear accumulation of endothelial nitric oxide synthase (eNOS), resulting in dilated retinal capillaries and pericytes via the nitric oxide synthase (NOS)/nitric oxide (NO)/guanosine 3',5'- monophosphate (cGMP) signalling pathway. Neuronal α7-nAChR activation directly relaxed retinal capillaries and pericytes via a neurovascular coupling mechanism. α7-nAChR also increased the vesicular release of GABA, possibly promoting the release of NO by binding to GABAA receptors in retinal ganglion cells (RGCs) and relaxing blood vessels via eNOS-NO, with GABA binding to GABAB receptors on retinal capillary endothelial cells. CONCLUSION AND IMPLICATIONS: α7-nAChR activation causes vasorelaxation of retinal capillaries.

Differential Modulation of the Excitatory and Inhibitory Synaptic Circuits of Retinal Ganglion Cells via Asiatic Acid in a Chronic Glaucoma Rat Model.

PURPOSE: To investigate whether asiatic acid (AA) can improve the quantity and function of retinal ganglion cells (RGCs), as well as how AA regulates synaptic pathways in rat models with chronic glaucoma. METHODS: In our study, a rat model of chronic glaucoma was prepared via the electrocoagulation of the episcleral veins. The numbers of surviving RGCs were counted via retrograde Fluorogold labeling, and a whole-cell patch clamp was used to clamp RGCs in normal retinal sections and in retinal sections 4 weeks after glaucoma induction. RESULTS: Retrograde-Fluorogold-labeled RGC loss caused by persistent glaucoma was decreased by AA. Additionally, AA reduced the postsynaptic current produced by N-methyl-D-aspartate (NMDA) and diminished miniature glutamatergic excitatory neurotransmission to RGCs. On the other hand, AA increased miniature gamma-aminobutyric acid (GABA)-ergic inhibitory neurotransmission to RGCs and enhanced the GABA-induced postsynaptic current. The excitability of the RGC itself was also decreased by AA. RGCs in glaucomatous slices were less excitable because AA decreased their spontaneous action potential frequency and membrane potential, which led to a hyperpolarized condition. CONCLUSIONS: AA directly protected RGCs in a chronic glaucoma rat model by lowering their hyperexcitability. To enhance RGCs' survival and function in glaucoma, AA may be a viable therapeutic drug.

Effect of Hypochlorous Acid on Blepharitis through Ultrasonic Atomization: A Randomized Clinical Trial.

PURPOSE: To evaluate the efficacy and safety of eyelid hygiene using topical 0.01% hypochlorous acid (HOCL) through ultrasonic atomization after 2 weeks in patients with blepharitis. DESIGN: Randomized controlled trial. METHODS: Patients with blepharitis were randomized into two groups: topical 0.01% HOCL through ultrasonic atomization (HOCL group, 42 eyes) or eyelid scrubs (control group, 37 eyes). Patients in both groups received warm compresses twice daily and topical 0.5% levofloxacin three times a day. Primary outcomes were the ocular surface disease index scores (OSDI), lid margin redness, lid margin abnormalities, meibum expressibility, meibum quality, and noninvasive breakup time after 2 weeks. Secondary outcomes were conjunctiva redness, corneal fluorescein staining, and tear meniscus height. A questionnaire of treatment adherence with a free response section was administered to confirm patient compliance and comments. RESULTS: Sixty-seven participants participated in this study. Both groups show an improvement in all primary outcomes, while statistically significant improvements in OSDI, lid margin redness, lid margin abnormality, meibum expressibility and quality are only limited to the HOCL group after 2 weeks of treatment (p < 0.05, p < 0.05, p < 0.001, p < 0.001 and p < 0.001, respectively). Subgroup analysis in HOCL reveals that only the change in lid margin abnormality and meibum expressibility in the mild-moderate meibomian glands loss patients at baseline has a statistically significant difference p < 0.05). Multiple linear regression shows that the improvement in OSDI is negatively associated with meibum expressibility score at the baseline (95% CI [-28.846, -1.815], p = 0.028). The patient compliance is 7.1 ± 2.0 in the HOCL group and 7.1 ± 1.8 in the control group (p > 0.05). No adverse events are reported. CONCLUSION: Topical 0.01% HOCL through ultrasonic atomization is a tolerable and effective eyelid hygiene treatment for blepharitis.

Biological Immune Mechanism of Retina.

The blood-retinal barrier (BRB) is a well-recognized mechanism that underlies the retina's immunological privilege. The BRB is formed locally by inhibitory molecules that bind to cell membranes, as well as by the suppression of systemic immune responses. Recent studies have revealed that microglial cells are essential for maintaining immunological privilege within the retina by regulating the immune response. They achieve this by enhancing or reducing ocular inflammation. Furthermore, retinal pigment epithelium (RPE) regulates the behavior of immune cells within the retina, which can lead microglial cells to reduce inflammation and promote immunological tolerance. With the aim of better understanding the biology of immunological processes within the retina, this article reviews the BRB and discusses the factors, systemic immune responses, microglia, RPE, and their associated enzymes that enable the BRB.

Gut microbiota and derived metabolomic profiling in glaucoma with progressive neurodegeneration.

Glaucoma is a multifactorial, neurodegenerative disorder characterized by the loss of retinal ganglion cells (RGCs). Crosstalk between the gut microbiota and host is involved in the progression of many neurodegenerative diseases, although little is known about its role in glaucoma. To investigated the alterations of the gut microbiota and derived metabolites in glaucomatous rats, and the interaction with RGCs, we performed 16S rRNA (V1-V9) sequencing and untargeted metabolomic analyses. The microbial composition differed significantly between the two groups, and the diversity of cecal bacteria was dramatically reduced in glaucomatous rats. The Firmicutes/Bacteroidetes (F/B) ratio, Verrucomicrobia phylum, and some bacterial genera (Romboutsia, Akkermansia, and Bacteroides) were dramatically increased in the glaucomatous rat model compared with the control, which showed negative correlation with RGCs. Untargeted metabolomic analysis identified 284 differentially expressed metabolites, and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed considerable enrichment mainly in bile secretion pathways. The relationships among the metabolites enriched in the bile secretion pathway, differentially expressed cecal microbiota, and RGCs were investigated, and glutathione (GSH) was found to be negatively correlated with Bacteroides and F/B and positively correlated with RGCs. Reduced GSH level in the blood of glaucoma rats is further established, and was negatively correlated with Romboutsia and the F/B ratio and positively correlated with RGCs. This finding suggests the potential role of the gut microbiota and derived metabolites in glaucoma, and GSH, a major antioxidant metabolite, was related to their effects, indicating the potential for the development of gut microbiota-targeted interventions for glaucoma.

Activation of the GABA-alpha receptor by berberine rescues retinal ganglion cells to attenuate experimental diabetic retinopathy.

Purpose: The aim of this study was to investigate the role and mechanism of berberine (BBR) in the protection of injured retinal ganglion cells (RGCs) in diabetic retinopathy (DR). Methods: Experimental diabetic retinopathy rat model was successfully induced by a single intraperitoneal injection of streptozotocin (STZ, 60 mg/kg) in male SD rats with sufficient food and water for 8 weeks. Animals were randomly divided into four groups: (1) non-diabetic, (2) diabetic, (3) diabetic + BBR + PBS, and (4) diabetic + BBR + SR95531. BBR (100 mg/kg) was given daily by gavage to rats in the group (3) and group (4) for 8 weeks, and weekly intravitreal injections were conducted to rats in the group (3) with 5 µL of 1×PBS and rats in the group (4) with 5 µL of GABA-alpha receptor antagonist SR95531 to investigate the underlying mechanisms. The survival and apoptosis of RGCs were observed by fluorescence gold labeling technology and TUNEL staining. Visual function was evaluated by visual electrophysiological examination. Western blotting and immunofluorescence staining were used to analyze the expression of GABA-alpha receptors in RGCs. Results: In an animal model, BBR can increase the survival of RGCs, reduce RGCs apoptosis, and significantly improve the visual function. The reduction of GABA, PKC-α, and Bcl-2 protein expression caused by DR can be considerably increased by BBR. SR95531 inhibits BBR's protective effect on RGC and visual function, as well as its upregulation of PKC-α and Bcl-2. Conclusion: BBR is a promising preventive or adjuvant treatment for DR complications, and its key protective effect may involve the regulation of RGC apoptosis through the GABA-alpha receptor/protein kinase C-alpha (GABAAR/PKC-α) pathway.

Rapid and quantitative detection of tear MMP-9 for dry eye patients using a novel silicon nanowire-based biosensor.

Dry eye disease (DED) is the most common chronic eye disease characterized by ocular surface inflammation that affects hundreds of millions of people worldwide. The diagnosis and monitoring of DED require fast and reliable tools in the clinical setting. Matrix metalloproteinase 9 (MMP-9) has been proven to be a reliable indicator of DED owing to its close relationship with inflammation. A novel biosensor based on silicon nanowire-based field-effect transistor (SiNW FET) devices was fabricated for the quantitative measurement of MMP-9 in human tears. A modified controllable process was applied to improve the uniformity of the SiNWs in size and stabilize their performance with optical calibration at low salt concentrations for clinical application. With this protocol, correlation analysis proved the high agreement between the biosensor and enzyme-linked immunosorbent assay (correlation coefficient of 0.92 for DED patients and 0.90 for healthy controls). A diagnostic sensitivity of 86.96% and specificity of 90% were achieved in human tear samples from DED patients and healthy subjects in real-world clinical settings. Furthermore, the tear MMP-9 concentrations monitored using the device correlated with the therapeutic response of the patients with DED. Our enhanced SiNW biosensor device demonstrated its potential as an alternative tool for real-time diagnosis and monitoring for prognostic prediction toward point-of-care testing for DED.

Morphological and Functional Changes of Meibomian Glands in Pediatric and Adult Patients with Allergic Conjunctivitis.

Allergic conjunctivitis (AC) is one of the most common ocular disorders in clinical practice and is associated with meibomian gland dysfunction. This study aimed to explore the morphological and functional changes of meibomian glands (MGs) in pediatric and adult patients with AC and to analyze their potential predictors. In our prospective, observational cohort study, a total of 59 patients with AC were enrolled, with 30 patients aged ≤16 years in the pediatric group and 29 patients in the adult group. All patients underwent examinations at baseline and last visit when the complete resolution of conjunctival papillae was identified. An automatic MG analyzer was used to measure the morphological and functional parameters of MGs, including their area ratio (GA), tortuosity index (TI), and signal index (SI). Two groups were comparable at baseline in terms of characteristics and MG parameters (p > 0.05). The morphological (length, square, and GA) and functional MG parameters (SI) of AC patients significantly improved in the pediatric group after treatment (all p < 0.05), but not in the adult group. The change in the GA correlated with age, sex, GA, TI, and SI at baseline (all p < 0.05). Age (p = 0.001) and GA (p < 0.001) at baseline were predictors of an improvement in the GA of MGs. The findings showed that the structure and function of MGs in pediatric patients with AC seem to improve after the conjunctival papillae disappear, but not in adult patients.

Design of a Novel Fab-Like Antibody Fragment with Enhanced Stability and Affinity for Clinical use.

With increasing interest in applying recombinant monoclonal antibodies (mAbs) in human medicine, engineered mAb fragments with reduced size and improved stability are in demand to overcome current limitations in clinical use. Herein, a novel Fab-like antibody fragment generated via an in silico-based engineering approach where the CH1 and CL domains of Fab are replaced by the IgG1 CH3 domains is described. This construct, designated as FabCH3, maintains the natural N-terminus and C-terminus of IgG antibody, can be expressed at a high level in bacterial cells and, importantly, exhibits much higher stability and affinity than the parental Fab when tested in a mesothelin-specific Fab m912, as well as a vascular endothelial growth factor A (VEGFA)-specific Fab Ranibizumab (in vivo). The high-resolution crystal structures of m912 FabCH3 and m912 Fab are determined, and the comparative analysis reveals more rigid structures in both constant domains and complementarity-determining regions of FabCH3, explaining its enhanced stability and affinity. Overall, the stabilized FabCH3 described in this report provides a versatile platform for engineering Fab-like antibody fragments with higher stability and antigen-binding affinity that can be used as a distinct class of antibody therapeutics.

Alterations in the Retinal Vascular Network and Structure in Myelin Oligodendrocyte Glycoprotein Antibody-Associated Optic Neuritis: A Longitudinal OCTA Study.

PURPOSE: To investigate the longitudinal microstructural and microvascular changes in patients with myelin oligodendrocyte glycoprotein antibody-associated optic neuritis (MOG-ON) without new attacks. METHODS: We included 20 eyes of 12 MOG-ON patients without new attacks during the follow-up and 24 eyes of 12 age- and sex-matched healthy controls. RESULTS: The BCVA, retinal vessels and structure were significantly lower in MOG-ON eyes than in healthy eyes(all P < .05). In MOG-ON eyes, the BCVA (p = .408) and mean deviation (p = .854) were not significantly decreased at the follow-up visit. However, there were small, significant decreases in parafoveal vessel density (p = .026), peripapillary vessel density (p = .008), and RNFL thickness (p = .03), but not GCIPL thickness (p = .107). CONCLUSIONS: Ongoing deterioration was observed in RNFL thickness and parafoveal and peripapillary vessel density, but not GCIPL thinning, in MOG-ON eyes without a new attack of ON.

WAY-100635 Alleviates Corneal Lesions Through 5-HT1A Receptor-ROS-Autophagy Axis in Dry Eye.

Purpose: To explore whether 5-HT1A receptors are involved in the dry eye disease (DED) mouse model and reveal its underlying mechanism. Methods: A C57BL/6J mouse DED model was established via the administration of 0.2% benzalkonium chloride twice a day for 14 days. Corneal fluorescein sodium staining score and Schirmer I test were checked before, and on days 7, 14, and 21 after treatment. The experiment was randomly divided into control, DED, 5-HT1A receptor agonist with or without N-acetylcysteine (NAC) and 5-HT1A receptor antagonist with or without NAC groups. The mRNA expression of inflammatory cytokines was measured by reverse transcription-quantitative polymerase chain reaction. Cellular reactive oxygen species (ROS) were detected by 2', 7'-dichlorodihydrofluorescein diacetate assays. Western blot analysis was used to measure the expression levels of autophagic proteins microtubule-associated protein 1 light chain 3 (LC3B-I/II) and autophagy-related gene 5 (ATG5). Results: 5-HT1A receptor agonist (8-OH-DPAT) increased corneal fluorescein sodium staining spots and 5-HT1A receptor antagonist (WAY-100635) decreased them. Treatment with 8-OH-DPAT was associated with the gene expression of more inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), C-C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 10 (CXCL10) compared with treatment with WAY-100635. An increased expression of LC3B-I/II and ATG5 was observed in corneal epithelial cells in the mouse model of DED. 8-OH-DPAT significantly enhanced the expression of LC3B-I/II and ATG5 by disrupting ROS levels. WAY-100635 alleviates autophagy by inhibiting ROS production. Conclusion: Excessive ROS release through 8-OH-DPAT induction can lead to impaired autophagy and increased inflammatory response in DED. WAY-100635 reduces corneal epithelial defects and inflammation in DED, as well as alleviates autophagy by inhibiting ROS production. The activation of the 5-HT1A receptor-ROS-autophagy axis is critically involved in DED development.

Long Non-coding RNAs Gabarapl2 and Chrnb2 Positively Regulate Inflammatory Signaling in a Mouse Model of Dry Eye.

Purpose: To elucidate the expression profile and the potential role of long non-coding ribonucleic acids (RNAs) (lncRNAs) in a dry eye disease (DED) model. Methods: A DED model was established in C57BL/6J mice with 0.2% benzalkonium chloride (BAC) twice a day for 14 days. The differentially expressed lncRNAs were detected by RNA-seq technology (Gene Expression Omnibus, GEO GSE186450) and the aberrantly expressed lncRNAs were further verified by RT-qPCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to predicate the related candidate genes and potential pathological pathways. Cells from a human corneal epithelial cell line (HCECs) were cultured under hyperosmolarity. The regulation of inflammatory factors by silencing potential targeted lncRNAs was verified in vitro in HCECs. Results: In our study, a significant increase in corneal fluorescence staining and a reduction in tear production were observed in DED mice at all follow-ups compared with the controls, and the differences were increasing over time. In total, 2,649 upregulated and 704 downregulated lncRNAs were identified in DED mice. We selected six aberrantly expressed and most abundant lncRNAs and performed RT-qPCR using the samples for RNA-seq. Chrnb2, Gabarapl2, and Usp31 were thereby confirmed as the most significantly altered lncRNAs. Pathway analysis revealed that the neuroactive ligand-receptor interaction signaling pathway was the most enriched, followed by the calcium signaling pathway and cytokine-cytokine receptor interaction. Following treatment of Gabarapl2 siRNA and Chrnb2 siRNA, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 were significantly downregulated in the HCECs. Conclusion: Our study suggests that Chrnb2 and Gabarapl2 may be involved in the inflammation response by regulating TNF-α, IL-1ß, and IL-6 in DED. These candidate lncRNAs may be both potential biomarkers and therapeutic targets for DED.